In the STED microscope (Hell and Wichmann U.S. Pat. No. 5,731,588, Baer, U.S. Pat. No. 5,866,911), the resolution of a scanning fluorescent microscope is improved by scanning the spot of excitation light synchronously and concentrically with a doughnut-shaped beam of a wavelength able to quench the fluorescent excitation by stimulated emission. Because near the center of the doughnut-shaped beam, the intensity increases with distance from the center, the quenching also increases with such distance, selectively quenching the periphery of the scanned spot, decreasing its size and thereby increasing the resolution of the microscope. In the form of STED microscope in which the doughnut-shaped beam has a central point of substantially zero intensity (Baer U.S. Pat. No. 5,866,911), by increasing the power of that beam, the quenching can reach any desired level at arbitrary closeness to the central point, without quenching fluorescence at the central point. This leads to resolution limited effectively only by the ability to generate the high powers of the doughnut-shaped beam and the ability of the specimen to tolerate the high powers. Since that first proposed use of a zero-centered doughnut-shaped beam in microscopy, several other microscopy techniques have been proposed using such beams.
Because in microscopy applications of such beams, the optimum wavelength of the quenching beam depends on the fluorescent dye or protein, it is desirable to have a method for forming such beams where the wavelength can be changed, sometimes very rapidly, while preserving the zero-intensity of the beam central point. Also, it is sometimes desirable to make such doughnut shaped beams with light of very short pulses that have a relatively broad spectral distribution. Finally, to improve the quality of the doughnut beam produced, it may be desirable to locate the doughnut beam making means as close as possible to the microscope objective lens as possible, in which case the fluorescent light emitted from the specimen would have to pass through said means in addition to both the excitation and quenching beams, and each of these bands of light would in general have different wavelengths. Therefore, ideally, the method for making the zero-centered doughnut-shaped beams would be invariant with respect to the wavelength.
Several methods have been proposed to address the problem of making zero-centered doughnut beams that can operate at more than one wavelength. Baer (PCT #US06/01959) proposed creating the doughnut pattern by means of a pyramid of glass triangles, in conjunction with a four-quadrant reflector. By adjusting the various phase retardations by tilting of the pyramid and translation of the reflective quadrants, the device can be corrected for two chosen wavelengths, and is approximately correct for the wavelengths between these two. However, because in this application, phase errors of even a small fraction of a wavelength can be detrimental to the performance of the system, such correction is not always adequate, particularly over a large wavelength range.
Another proposed solution has been to use an electronically programmable Spatial Light Modulator “SLM” (Hamamatsu Photonics K. K.) which can be reprogrammed as the wavelength is changed. However this proposed solution involves introducing additional optics to direct the beam onto the SLM and pass the reflected beam to the rest of the system, and in some cases to expand the beam for presentation to the SLM and to contract it for presentation to the rest of the optical system, and such optics create an opportunity for scattering, spurious reflection and introduction of additional phase errors. Furthermore the system is relatively expensive, and cannot deal with extremely rapid changes in wavelength required in some microscope techniques or creation of zero-center patterns from an intrinsically broadband source.
Yet another solution has been to create a plurality of spiral phase plates on a common glass sheet backing, such that each separate phase plate is corrected for a specific wavelength, and the wavelengths for the individual plates differed by 20 nm, covering a chosen interval of the spectrum. However even when a given phase plate is used with the exact wavelength for which it had been designed, it is difficult to control the thickness of the think layer coatings with sufficient accuracy to create a perfect pattern with a substantially zero intensity center. Also it is impossible to quickly vary excitation wavelength because of the need to rotate the glass sheet to put another phase plate into position. Furthermore the system cannot work with a broadband source.
Finally, Gugel (U.S. Patent Appl 2006/0007535 A1) has proposed using the relative wavelength independent phase shift during total internal reflection, combined with the difference in phase shift depending on whether or not there is a metallic backing layer overlying the reflecting surface, to approximate an achromatic phase shift, between a beam portion reflected from a surface with a metallic backing layer compared to a beam portion reflected from a surface without such a metallic backing layer. However, with a metallic backing layer, the absorption does show a wavelength dependence, so that it is difficult or impossible to ensure cancellation at the central point over a wide range of wavelengths. Furthermore when such a system is used in a Mach-Zehnder interferometer to approximate a circular pattern with a zero intensity center, the light at two orthogonal axes is plane polarized, which may result in incomplete quenching in a STED microscope, thereby reducing resolution. Furthermore such a solution is bulky, and to avoid loss of alignment with even small temperature changes requires operation in a very limited temperature range.
Thus there remains an unfulfilled need for apparatus to create zero central point patterns that is compact and accurate over a wide range of wavelengths without requiring readjustment.